Caspase 1 gene transfer animal

ABSTRACT

A transgenic non-human mammal or its offspring which carries DNA having exogenous caspase 1 gene integrated thereinto in such a manner as to express the gene specifically to skin.  
     In view that the transgenic non-human mammal of the present invention spontaneously develops atopic dermatitis in the absence of any specific pathogenic microorganism, the mammal is useful as a disease model animal. Use of the transgenic animal of the present invention enables development of preventive and/or therapeutic drugs for atopic dermatitis based on natural immunity and to clarify the onset mechanism of atopic diseases.

TECHNICAL FIELD

[0001] The present invention relates to a transgenic animal useful as ananimal model for atopic dermatitis.

BACKGROUND ART

[0002] Atopic diseases, including atopic dermatitis, asthma, allergicrhinitis, and allergic conjuctivitis, are pathological conditions whichpermit the subject's own immune system to destroy, and cause disturbanceof organs as a result of hyper-responses to environmental antigens towhich healthy subjects usually exhibit no response. A conceivablemechanism of development of these diseases includes contribution of Th2cytokine, which promotes allergic responses. The induction mechanism andregulatory mechanism, which are crucial from the physiological andpharmacological viewpoints, have not yet been completely elucidated.

[0003] At present, mainstream treatments of atopic diseases includeavoidance of antigens; administration of antihistaminic agents whichserve as antagonists against binding of a mediator such as histamine toa receptor; and administration of anti-inflammatory steroids. However,development of improved therapies targeting more specific actionmechanisms remains hindered, because no suitable test animals have beenprovided.

[0004] According to conventional techniques, in order to elicit anallergic response from a test animal, the animal must first besensitized through repeated immunization of an antigen or an allergen inadvance, and then be challenged with the same type of antigen or theallergen. Such a process not only imposes considerable workload forsimultaneously sensitizing numerous animals, but also producesnon-uniformity between animals in terms of reactivity, raising potentialproblems on reproducibility of the test.

[0005] In recent years, NC/Nga mice have attracted attention for theirpotential use as an animal model of atopic dermatitis. However, thisdermatitis is developed only in the case where the host mouse carries amite, and further, the percent onset is inconsistent and symptomsdevelop in different forms.

[0006] Tests using animals are indispensable in the development ofremedies for atopic diseases, and in particular, demand exists foranimal models of atopic dermatitis. To date, however, no such animalmodels of atopic dermatitis having a well-established hereditarybackground and clarified immunological traits and being available forresearch and development of remedies and pharmaceuticals underconditions from which specific pathogenic microorganisms are eliminatedhave ever existed or put into practical use.

[0007] Accordingly, an object of the present invention is to create ananimal that is useful as an atopic dermatitis model.

DISCLOSURE OF THE INVENTION

[0008] In view of the foregoing, the present inventors have focusedtheir research efforts on interleukin 18 (IL-18). IL-18, when processedby a protease called caspase 1 (IL-1β converting enzyme), is transformedfrom its precursor form to a mature form. The functions of mature IL-18are known to include: (1) induction of production of IFN-γ, (2)promotion of Fas-mediated apoptosis through facilitated expression ofFas ligand, (3) induction of GM-CSF, and (4) inhibition of IgEproduction in the co-presence of IL-12. IL-18 is expressed not only inimmune tissue but also in a variety of tissues including osteoblast-likestromal cells, keratinocytes, small intestinal epithelial cells,adrenocortical cells, and hypophysis cells, and their physiologicalroles have been energetically studied (see, for example, Immunology,1997-98, published by Nakayama Shoten, 62-72; and Clinical Immunology,30(2), 191-198, 1998). Recently, the present inventors have discoveredthat over-expression of IL-18, when occurring alone, promotes productionof IL-4 and IL-13 and elicits production of IgE. This signifies closeinterrelation between IL-18 and Th2 cytokine, which relates to the onsetof atopic diseases. Therefore, the inventors have considered that if ananimal model having enhanced ability in secretion of caspase 1, whichconverts IL-18 to its mature form, can be created, such an animal wouldbe useful in elucidation of the onset mechanism of atopic diseases anddevelopment of therapeutic methods therefor. However, since caspase 1 isan apoptosis-inducing enzyme, simple transfer of a caspase 1 gene forexpression in a living body is lethal to the host. Moreover, in order tocause a host to secrete mature IL-18, expression of caspase 1 in cellsthat produce pro-IL-18 is necessary. Therefore, the inventors haveconsidered that if recombinant DNA engineered so as to express caspase 1gene specifically in the skin capable of producing pro-IL-18 istransferred to animal cells, the host animal continuously secretesmature IL-18 in blood, and when such an animal is raised underconditions free from specific pathogenic microorganisms such as mitesand molds, transgenic animals showing symptoms of atopic dermatitis canbe created, thus leading to completion of the invention. In thisconnection, the present inventors previously studied apoptosis andinflammation due to caspase 1, through direct injection of caspase 1gene to a healthy mouse, rather than to a fertilized egg thereof (J.Dermatol. Sci. 21(1999): 49-58). However, this mouse did not developspontaneous inflammation, and thus completely differs from the animal ofthe present invention.

[0009] Thus, the present invention provides a transgenic non-humanmammal or its offspring, comprising DNA engineered to contain exogenouscaspase 1 gene in such a manner as to be specifically expressed in theskin, and a method for creating the same.

[0010] The present invention also provides a method for screeningpreventive and/or therapeutic substances for atopic dermatitis,characterized by administering a test substance to the mentionedtransgenic animal and investigating the ameliorating effect of thesubstance on atopic dermatitis; and by use of the screening method, toprovide a preventive or therapeutic drug for atopic dermatitis which isdetermined to have ameliorating effect for atopic dermatitis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 schematically shows arrangement of components of therecombinant DNA employed in gene transfer;

[0012]FIG. 2 shows the results of Northern blot analysis regardingexpression of mRNA of hproCASP1 in various tissues from KCASP1Tg;

[0013]FIG. 3 shows the results of immunoblotting using anti-hCASPantibody for expression of hCASP1 protein in the skin of KCASP1Tg;

[0014]FIG. 4 shows concentrations of IL-18, IL-1β, and IL-1α in sera ofKCASP1Tg;

[0015]FIG. 5 shows the frequency of skin scratching of KCASP1Tg andhealthy mice;

[0016]FIG. 6 shows time-course changes in blood IgE and histamine levelsof KCASP1Tg; and

[0017]FIG. 7 shows blood IgE levels of 14-week-old KCASP1Tg and healthymice.

BEST MODE FOR CARRYING OUT THE INVENTION

[0018] The somatic cells and germ cells of the transgenic animal of thepresent invention harbor DNA in which exogenous caspase 1 (CASP1) geneis integrated so that the gene is skin-specifically expressed. Theexogenous caspase 1 gene is preferably derived from humans or mice, andexamples include human procaspase 1 (hproCASP1) gene, murine procaspase1 (mproCASP1) gene, human caspase 1 (hCASP1) gene, and murine caspase 1(mCASP1) gene, with 1.4 kb cDNA of a full coding region of hproCASP1being particularly preferred.

[0019] Preferably, DNA in which the exogenous caspase 1 gene has beenintegrated so that the gene is skin-specifically expressed is arecombinant DNA containing a exogenous caspase 1 gene and a promoter forskin-specific protein. Examples of the promoter for a skin-specificprotein include promoters for proteins which are specifically found inthe skin, and specific examples include promoters for keratin 14,keratin 5, keratin 1, keratin 10, and involcrin, with keratin promotersbeing particularly preferred. The exogenous caspase 1 gene is linked tothe downstream of the promoter of the skin-specific protein, and inorder to improve expression efficiency of the gene, preferably, anintron, such as β-globin intron, is also linked.

[0020] Preferably, the above-mentioned DNA contains a sequence of polyA(usually called a terminator) for terminating transcription of messengerRNA of interest in a transgenic mammal, for enabling manipulation ofgene expression by use of gene sequences derived from, for example,viruses and mammals. Preferably, polyA of the aforementionedskin-specific protein is employed, and more preferably, polyA of keratinis employed. In addition, in order to attain enhanced expression of thegene of interest, a splicing signal or an enhancer region of the gene,or a portion of an intron of eukaryotic gene may be ligated to the 5′upstream of the promoter region, between the promoter region and thetranslational region, or the 3′ downstream of the translational region,depending on needs.

[0021] Examples of the non-human mammals for transferring thethus-obtained recombinant DNA include cows, pigs, sheep, goats, rabbits,dogs, cats, guinea pigs, hamsters, rats, and mice. Of these mammals,rabbits, dogs, cats, guinea pigs, hamsters, mice, and rats arepreferred. Rodentia; in particular, mice, are particularly preferred.

[0022] The transgenic animal of the present invention is created bytransferring DNA in which the aforementioned exogenous caspase 1 genehas been integrated so that the gene is expressed specifically in theskin to, for example, a fertilized egg of a non-human mammal, and thenthe egg is implanted into a female individual of the mammal. Preferably,the fertilized egg to be employed is in the period of male pronucleus(approximately 12 hours after fertilization). Examples of techniques fortransferring the recombinant DNA include the calcium phosphate method,electric pulse application, lipofection, agglutination, microinjection,the particle gun method, and DEAE-dextran method, with microinjectionbeing particularly preferred.

[0023] The fertilized egg to which the recombinant DNA has beenincorporated is implanted to a female animal of the same species of theanimal from which the egg has been obtained. Preferably, implantation iscarried out artificially; i.e., through transplantation and implantationto the oviduct of a pseudopregnant female animal. From the offspringproduced by the fertilized-egg-implanted animal, individuals expressingthe gene of interest are selected and the selected individuals can bereproduced over generations.

[0024] Whether or not the obtained transgenic animal bears the gene ofinterest can be confirmed by collecting DNA from a skin sample andsubjecting the sample to polymerase chain reaction (PCR) and Southernblotting for analysis of the transferred gene.

[0025] The thus-obtained transgenic animal of the present inventionexpresses exogenous caspase 1 gene in the skin, and manifests symptomsof atopic dermatitis even under the absence of a specific pathogenicmicroorganism. Another characteristic feature of the animal of thepresent invention is prolonged life span.

[0026] The transgenic animal of the present invention has exogenouscaspase 1 gene only in the skin. In other words, the exogenous caspase 1gene is not contained in other tissues of, for example, the liver,kidneys, lungs, brain, or spleen. Thus, the skin of the transgenicanimal of the present invention contains mature IL-18 and mature IL-1β.Moreover, the blood of the transgenic animal of the present inventioncontains both mature IL-18 and mature IL-1β in greater amounts ascompared with healthy animals, with the mature IL-18 content beinghigher relative to mature IL-1β.

[0027] The transgenic animal of the present invention develops symptomsof atopic dermatitis—for example, rodent dermatitis, erosive dermatitis,skin ulcer, and erosion—from 8 weeks of age or thereabouts. From 10weeks of age or thereabouts, observation of skin tissue under an opticalmicroscope will reveal psoriasis-like changes accompanying parakeratosisor other changes such as acanthosisin circumferential thick epidermalportions around ulceration, and the corium of the ulcerate portion isinfiltrated with monocytes and mast cells. The keratinocytes of thelesion show acidophilic necrosis which accompanies pycnosis, indicatingapoptosis of keratinocytes. Moreover, the transgenic animals createdaccording to the present invention scratch their skin much morefrequently than do healthy counterparts, thereby confirming strongitchiness unique to atopic dermatitis. The transgenic animals of thepresent invention manifest a symptom characteristic to atopicdermatitis; i.e., high blood histamine level and high IgE level.

[0028] As described above, since the transgenic animals of the presentinvention manifest symptoms of atopic dermatitis in the absence ofspecific pathogenic microorganisms, they are useful as an animal modelof atopic dermatitis. That is, administration of a test substance to thetransgenic animal of the present invention or its offspring andverification of ameliorating effect on atopic dermatitis enablesscreening of preventive and/or therapeutic substances for atopicdermatitis. The ameliorating effect on atopic dermatitis may be verifiedthrough measurement of blood mature IL-18 level, detection of matureIL-18 in the skin, visual observation, or observation of skin tissueunder a microscope, solely or in appropriate combination. Testsubstances whose effect of ameliorating atopic dermatitis have beenconfirmed through screening are useful as preventive and/or therapeuticdrugs for atopic dermatitis.

EXAMPLES

[0029] The present invention will next be described in more detail byway of examples, which should not be construed as limiting theinvention.

[0030] The test methods and materials employed in the Example describedbelow are as follows:

[0031] (1) Northern Blotting

[0032] Murine caspase 1 (mCASP1) cDNA (Cell, 75, 653-660 (1993)) wasprocured from Osaka University (Dr. Miura). Total RNA, in its entirety,was obtained through extraction by use of an Isogen reagent (product ofNippon Gene Co., Ltd.) from tissues of CASP1 transgenic mice (KCASP1Tg)(obtained in Example 1 described hereinbelow) and control mice. InNorthern blotting analysis, 10 μg of total RNA were size-fractionated byelectrophoresis on 2% formaldehyde/agarose gels. RNA was transferredonto a nylon membrane (Immobilon-N, product of Millipore Co.) and probedwith ³²P-labeled cDNAs corresponding to human and murine CASP1(regarding human CASP1 cDNA, see J. Dermatol. Sci., 21 (1994), 49-58).The RNA and cDNA were hybridized on the membrane. Subsequently, theblots on the membrane were washed twice with 1×SSC/0.1% SDS at 42° C.,and twice with 2×SSC/0.1% SDS, and then exposed onto X-ray film at −70°C.

[0033] (2) Cytokines, Cytokine Assays, and Antibodies

[0034] Recombinant murine IL-1β (rmIL-1β) was purchased from R&D System.Human pro-IL-1β (hproIL-1β) was prepared in accordance with the methoddescribed in J. Exp. Med. 174: 821 (1991). Recombinant human IL-1β(rhIL-1β) and anti-human IL1β antibody were provided by OtsukaPharmaceuticals. IL-1α level was measured by use of an ELISA kit(product of Endogen), and IL-1β and IFN-γ levels were measured by use ofa kit manufactured by R&D System. Biological activities of IL-18 weredetermined through measurement of IFN-γ inducing activities by use ofIL-18-responsive murine NK cells. Recombinant murine IL-18 (rmIL-18),rabbit neutralizing anti-murine IL-18 antibody and murine IL-18 ELISAkits were provided by Hayashibara Biochemical Laboratories. Use of themurine IL-18 ELISA kit permitted detection of IL-18 within a range of 10to 1,000 pg/mL.

[0035] (3) Immunohistochemistry

[0036] Biopsy specimens obtained from transgenic mice and wild-type micewere fixed with phosphate-buffered formalin for two hours. Subsequently,the specimens were prepared for cutting of paraffin sections. Thesamples were immediately frozen in OCT compound (product of Miles) whichis an embedding material for frozen tissues, and stored at −70° C.Cryostat sections (5 μm) were fixed with acetone for 5 minutes at 4° C.An appropriately diluted primary antibody was added to samples forincubation for one hour. After washing of the samples, the primaryantibodies that had been bound were visualized by use of a VectasteinElite kit (product of Vector Laboratories) with AEC (product of DakoJapan) as a substrate.

[0037] (4) Terminal Deoxynucleotidyl Transferase-Mediated dUTP-BiotinNick End Labeling (TUNEL)

[0038] DNA fragmentation of skin-biopsy specimens in paraffin sectionswere examined by TUNEL staining in accordance with the method describedin Arch. Dermatol., 133: 845 (1997).

[0039] (5) Immunoblotting

[0040] Immunoblotting was performed in accordance with the methoddescribed in J. Clin. Invest. 87: 1066 (1991). DNA and RNA were removedby use of an Isogen kit. Epidermal cell lysates prepared from transgenicmice and control mice were individually suspended in SDS-sample bufferunder reducing conditions. Then, the electrophoresed proteins weretransferred onto a nitrocellulose membrane (product of Scheicher &Schuell) by use of a semi-dry blotter (product of Bio-Rad). A primaryantibody was added to the membrane for incubation for one hour.Subsequently, alkaline-phosphatase-labeled anti-mouse IgG or anti-rabbitIgG was added to the membrane for secondary incubation. Finally, thesamples were allowed to develop color with a Western Blue Substrate(product of Promega).

[0041] (6) Pro-IL-1β (ProIL-1β) Processing Activity

[0042] Recombinant human pro-IL-1β (rhproIL-1β) was incubated along withfresh epidermal lysate for 10 to 30 minutes. SDS-sample buffer was addedthereto, and the mixture was immediately boiled. The resultant sampleswere analyzed by immunoblotting by use of an anti-human IL-1β antibodyand a Western Blue detection kit (product of Promega).

Example 1

[0043] (1) DNA Construct and Creation of a Transgenic Mice

[0044] 1.4 kb cDNA encoding the entire coding region of hproCASP1 wasligated into human keratin 14 promoter (kindly offered by Dr. E. Fuchs,Chicago University; Nature, 374, 159-162 (1995)) and rabbit β-globinintron (kindly offered by Dr. Tanaka, Kyoto University; Nature, 374,159-162 (1995)) by blunt-end ligation (in FIG. 1, “K14 promoter,”“β-glob. Int.,” “1.4 kb hproCASP1,” and “K14 polyA” refer to humankeratin 14 promoter, rabbit β-globin intron, 1.4 kb of human procaspase1 cDNA, and human keratin 14 polyA, respectively). The thus-obtained DNAfragment was injected into fertilized eggs of C57BL/L6 mice (CharlesRiver Japan, Inc.) through microinjection in accordance with the methoddescribed in Dev. Growth Differ., 39: 257 (1997).

[0045] (2) Verification of Over-Expression of hCASP1 in the Skin ofTransgenic Mice

[0046] The offspring mice was screened for incorporation of thetransgene by PCR and Southern blotting using DNA from the tail skin. Ofa total of 96 newborn mice, four mice (male 3, female 1) were hCASP1transgenic (hereinafter these mice will be abbreviated as KCASP1Tg). TheKCASP1Tg were healthy when born, and grew normally thereafter. However,before 8-weeks of age, they were smaller than their wild-typelittermates. After this point in time, KCASP1Tg manifested chronicactive dermatitis. Of the three male KCASP1Tg, one was mated withwild-type females, and generated KCASP1Tg and wild-type offspring in an1:1 male:female ratio. All experiments were performed on the lineheterozygous for the transgene compared with non-transgenic or wild-typelittermates.

[0047] (3) Skin Conditions

[0048] Under conditions in which no special pathogens were detected,from 8 weeks of age, the KCASP1Tg clearly began to manifest rodentdermatitis of medium degree in peripheral portions of their eyes, whichdeveloped rapidly to grave erosive dermatitis. From time to time, skinulcers were observed. Within one to two weeks thereafter, these symptomsdeveloped on the face, ears, neck, torso, and legs. Subsequently,re-epitheliation occurred, and lichenoid dermatitis was locallydeveloped, with recurrence of erosion and ulcers. After 16 weeks, aplurality of foci of skin disease were formed, and the ears and eye lidswere deformed. Hair on the face and extremities disappeared, onlyleaving integument accompanied by multiple scars.

[0049] Under an optical microscope, the epidermis of the KCASP1Tg didnot exhibit particular histological changes up until 6 weeks of age. Thethick epidermis surrounding the ulcerated lesions of the KCASP1Tg of 10weeks of age presented psoriasis-like changes accompanying parakeratosisand dermatitis partially associated with acanthosis. The corium in theulcerated sites was infiltrated with numerous monocytes. Thekeratinocytes in the lesion sites exhibited eosinophilic necrosis withnuclear condensation, which is indicative of apoptosis of keratinocytes.In fact, the nuclei of the cells present in the skin lesion sitescontaining many keratinocytes were stained positive by means of TUNELstaining, which is employed for identification in DNA fragmentation. Incontrast, no TUNEL-positive keratinocytes were observed in the skinsamples from control littermates.

[0050] High level hCASP1 was detected in thick epidermal tissue samplesfrom KCASP1Tg. In contrast, samples from control mice tested negative.In order to investigate whether ulceration was attributed to thefunctional Fas ligand (Fas-L) induced by mature IL-18 processed fromendogenous pro-IL-18 by exogenous hCASP1, Fas-L neutralizing antibodywas administered to KCASP1Tg before they started to develop changes inthe skin. Specifically, in accordance with the method described in J.Exp. Med., 182: 1777 (1995), Fas-L neutralizing antibody (1 mg; MFL1,kindly provided by Dr. Higaki, Juntendo University) wasintraperitoneally injected to each KCASP1Tg every week from the 5th weekto the 9th week, and conditions of skin ulceration were observed.However, intensive treatment with anti-Fas-L neutralizing antibody wasuseless, permitting skin ulcers to develop. Therefore, apoptosis ofkeratinocytes is suggested to be induced independent of the action ofthe Fas/Fas-L pathway. IL-18 did not induce apoptosis in any sample fromthe tested cell lines and tested primary culture cells. Moreover,keratinocyte-specific IL-1 α transgenic mice did not manifestapoptosis-related skin ulcers. This suggests that IL-1 β does notparticipate in apoptosis of skin cells of KCASP1Tg, although IL-1β hasthe same functions as IL-1α.

[0051] (4) Detection of Mature hCASP1 in Skin

[0052] Presence of hproCASP1 mRNA in cells of ear epidermis, backepidermis, liver, kidney, colon, lung, brain, and spleen of KCASP1Tgwere detected through Northern blotting (FIG. 2). As a result, 1.4 kbhproCASP1 mRNA was found only in epidermal cells from the ear and theback of KCASP1Tg, but not in other tissues (liver, kidney, colon, lung,brain, and spleen of KCASP1Tg). FIG. 2 shows the results of Northernblotting: lane 1: a sample obtained from a non-transgenic littermates,lanes 2-9: hproCASP1 mRNA in the ear epidermal tissue, back epidermaltissue, liver, kidney, colon, lung, brain, and spleen of KCASP1Tg.

[0053] In order for caspase to exhibit its biological activities, properprocessing is generally required. Pro-CASP1 has been reported to undergoautoproteolysis in vitro to thereby form mature CASP1 (Molecular Cell,1:319 (1998); J. BIol. Chem., 271:13273 (1996)). The reports suggestthat KCASP1Tg contains hCASP1 which has been spontaneously activated inthe skin. In order to confirm this suggestion, the size of hCASP1protein in KCASP1Tg skin lysate was measured by use of anti-hCASP1antibody through immunoblotting (J. Biol. Chem., 271:13273 (1996)). Asshown in FIG. 3, the skin lysate contained two activated components ofhCASP1 (p20 and p10) and a precursor of p45, whereas the skin lysatefrom wild-type littermates contained no such component described above.These results reveal that hCASP1 in KCASP1Tg skin was spontaneouslycleaved into mature form. In FIG. 3, lane 1: positive control THP-1cells, lane 2: recombinant p20/p10 hCASP1, lane 3: KCASP1Tg skin lysate,lane 4: wild-type skin lysate.

[0054] Processing activity of hCASP1 in the skin of KCASP1Tg wasmeasured by use of pro-IL-1β as a substrate in vitro. KCASP1Tg epidermalcell lysate was found to have produced 17 kD mature fragments throughcleavage of 31 kD recombinant hproIL-1β. This cleavage was inhibited bya synthesized CASP1 inhibitor and iodoacetamide.

[0055] In addition, measurement was performed as to whether or nothCASP1 in the skin of KCASP1Tg exhibits biological activities in vitro.Since IL-1β and IL-18 were found to be constantly expressed inkeratinocytes through experiments making use of mRNA, the size of eachcytokine at the protein level in the skin was measured. Skin lysate fromKCASP1Tg contained both mature IL-18 and mature IL-1β, whereas skinlysate from wild-type contained no such component. This is becausetransfected hCASP1 spontaneously cleaves endogenous precursor-type IL-1βand precursor-type IL-18 through spontaneous activation.

[0056] (5) IL-18 Level in Blood

[0057] Measurement was performed as to whether or not local activationof IL-18 and IL-1β by exogenous hCASP1 induces systemic accumulation ofindividual mature cytokines. FIG. 4 shows a high serum IL-18 level inKCASP1Tg of four weeks of age, which is statistically significant. Theserum IL-18 level in wild-type littermates remained low (0.1 ng/mL orless) throughout the living period. The serum concentration of IL-18 inKCASP1Tgs gradually increased with growth. Only a small amount of IL-1βin KCASP1Tg serum was detected from the age of 12 weeks, whereas noIL-1β was detected in wild-type. Over-expression of hCASP1 inkeratinocytes selectively elevated serum concentration of IL-18 ratherthan IL-1β.

[0058] In order to verify that serum IL-18 of KCASP1Tg is mature IL-18,biological activities of KCASP1Tg serum IL-18 were investigated. As aresult, serum from KCASP1Tg was found to have ability to stimulateIL-18-responsive cloned natural killer cells to produce IFN-γ. Moreover,the ability to induce IFN-γ was completely inhibited by anti-IL-18antibody (neutralizing antibody). This indicates that the serum from aKCASP1Tg contains active IL-18. However, IFN-γ was not detected in serumfrom a KCASP1Tg in a normal state. Thus, KCASP1Tg continuously secretedmature IL-18 into circulating blood.

[0059] (6) Skin Scratching Behavior of Transgenic Mice

[0060] Incidence of skin scratching behavior of KCASP1Tgs and healthymice (C57BL/L6 mice) was measured for 40 minutes by visual observation.

[0061] As a result, as shown in FIG. 5, healthy mice scratched theirskin 50 times or less per 10 minutes, whereas KCASP1Tgs scratched 200times or more times per 10 minutes, thereby demonstrating production ofsevere examthemata accompanying itchiness.

[0062] (7) Blood IgE Level and Blood Histamine Level

[0063] Blood IgE level (RIA method) and blood histamine level (ELISAmethod) of KCASP1Tgs were measured with time. The results are shown inFIG. 6. Blood IgE level of KCASP1Tg (14 weeks of age) and healthy micewas measured, and the results are shown in FIG. 7. These results showthat the blood histamine level of the transgenic mice of the presentinvention has elevated as high as 2,000 nM at 13 to 14 weeks of age,whereas blood IgE level has also elevated extremely high; i.e., not lessthan 50 μg/mm at 16 weeks of age.

[0064] Industrial Applicability

[0065] Since the transgenic non-human mammals of the present inventionspontaneously develop atopic dermatitis in the absence of specificpathogenic microorganisms, they are useful as disease model animals. Useof transgenic animals of the present invention enables development ofpreventive/therapeutic drugs for atopic dermatitis on the mechanism ofnatural immunity, and elucidation of the onset mechanism of atopicdiseases.

1. A transgenic non-human mammal or its offspring comprising DNA havingexogenous caspase 1 gene integrated thereinto so that the gene isskin-specifically expressed.
 2. The transgenic non-human mammal or itsoffspring as recited in claim 1, wherein the DNA having exogenouscaspase 1 gene integrated thereinto so that the gene isskin-specifically expressed includes exogenous caspase 1 gene and apromoter for skin-specific protein.
 3. The transgenic non-human mammalor its offspring as recited in claim 2, wherein the promoter forskin-specific protein is a keratin promoter.
 4. The transgenic non-humanmammal or its offspring as recited in any one of claims 1 through 3,which continuously generates atopic dermatitis.
 5. The transgenicnon-human mammal or its offspring as recited in any one of claims 1through 4, which continuously secretes mature IL-18 to blood.
 6. Amethod for screening preventive and/or therapeutic substances comprisingadministering a test substance to an animal or its offspring as recitedin any of claims 1 through 5, for investigation of ameliorating effecton atopic dermatitis.
 7. A preventive and/or therapeutic substance foratopic dermatitis comprising a substance which is determined to exhibitan ameriolating effect on atopic dermatitis by employment of thescreening method as recited in claim
 6. 8. A method for creating ananimal or its offspring as recited in any of claims 1 through 5,comprising incorporating, to a fertilized egg of a non-human mammal, DNAin which exogenous caspase 1 gene has been transferred so that the geneis skin-specifically expressed, and implanting the fertilized egg to afemale individual of the mammal.